Periodic Reporting for period 1 - METAFRAX (Unconventional NMDAR signaling theory for Fragile X Syndrome)
Reporting period: 2021-02-01 to 2023-01-31
The METAFRAX project aims at developing the seeds for new pharmacotherapeutic strategies for neurological disorders associated with intellectual disability and autism. Fragile X syndrome is the most common cause of inherited intellectual disability, and the leading known genetic cause of autism.
This dominant phenotype represents a huge hindrance for clinicians in the treatment of Fragile X syndrome. Indeed, at present, there are no mechanism-based therapies for intellectual disability and autism. The main obstacle has been to identify the defective cellular processes in the brain that disrupt behavior and cognition.
Earlier work on monogenic animal models of Fragile X syndrome supported the idea that one axis of pathophysiology in the syndrome is synaptic plasticity and NMDA receptor-mediated protein synthesis (NMDAR). Moreover, it has recently emerged that NMDAR can signal in a non-conventional manner, but this new mode of operation has yet to be characterized.
In this line, the first phase of this METAFRAX project pretended to develop the following key idea: when dysregulated, unconventional NMDAR signaling play a key role in the synaptic physiopathology of intellectual disability and autism associated with Fragile X syndrome.
The first objective was to understand how unconventional NMDRs drive synaptic plasticity, by characterizing a new signaling cascade downstream non-ionotropic NMDAR. The second objective was to determine if this is dysregulated in the mouse model of Fragile X syndrome.
Overall, this project provides the basis for the future discovery of therapeutic targets for Fragile X syndrome and other cognitively impaired conditions.
This dominant phenotype represents a huge hindrance for clinicians in the treatment of Fragile X syndrome. Indeed, at present, there are no mechanism-based therapies for intellectual disability and autism. The main obstacle has been to identify the defective cellular processes in the brain that disrupt behavior and cognition.
Earlier work on monogenic animal models of Fragile X syndrome supported the idea that one axis of pathophysiology in the syndrome is synaptic plasticity and NMDA receptor-mediated protein synthesis (NMDAR). Moreover, it has recently emerged that NMDAR can signal in a non-conventional manner, but this new mode of operation has yet to be characterized.
In this line, the first phase of this METAFRAX project pretended to develop the following key idea: when dysregulated, unconventional NMDAR signaling play a key role in the synaptic physiopathology of intellectual disability and autism associated with Fragile X syndrome.
The first objective was to understand how unconventional NMDRs drive synaptic plasticity, by characterizing a new signaling cascade downstream non-ionotropic NMDAR. The second objective was to determine if this is dysregulated in the mouse model of Fragile X syndrome.
Overall, this project provides the basis for the future discovery of therapeutic targets for Fragile X syndrome and other cognitively impaired conditions.
The first step towards the achievement of these goals was to understand the role of non-conventional NMDARs in synaptic function, and more specifically in synaptic plasticity. For that, we first studied the role of NMDARs in specific forms of long-term synaptic plasticity, depending on their synaptic localization. To this end, we have developed and initiated the characterization of a transgenic mouse line, that were invalidated for the gene encoding the necessary subunit of the NMDAR in excitatory cells. Using the quadruple patch-clamp recording technique coupled to 2-photon laser-scanning microscopy, we discovered that presynaptic NMDARs were necessary for the induction of a specific form of synaptic plasticity, namely timing-dependent long-term depression, whereas postsynaptic NMDARs were necessary for the “opposite” form of plasticity, namely timing-dependent long-term potentiation. In addition, these mouse lines also enabled us to establish that NMDARs play a crucial role in neuronal morphology, and in particular in the maintenance and plasticity of dendritic spines.
Then, using pharmacological approaches, we probed for the mode of signalling of involved in these two forms of synaptic plasticity. We revealed that timing-dependent long-term depression relies on non-ionotropic presynaptic NMDAR signalling (Figure Summary) whereas timing-dependent long-term potentiation relies on ionotropic postsynaptic NMDAR signalling. We have then deciphered further the signalling cascade mediating timing-dependent long-term depression. Using transgenic lines combined with pharmacology, we showed that non-ionotropic NMDARs determine timing-dependent long-term depression by recruiting the presynaptic JNK2 protein, which interacts with Syntaxin1a (Figure Summary). We have also demonstrated that this signalling pathway is required for timing-dependent long-term depression, regardless of the neuronal firing frequency during the induction (Figure Summary). Overall, our results show that NMDARs may have a specific mode of operation, depending on their synaptic localization as well as their signaling mode, to regulate specific forms of synaptic plasticity.
We now aim at testing whether these mechanisms are dysregulated in mouse model for Fragile X syndrome. In this line, we have started to study the main mouse model for Fragile X syndrome, the Fmr1 KO mouse line. Characterization is ongoing, and our preliminary data on synaptic plasticity have not led to any significant conclusions. Another step is to verify whether the signalling pathways downstream non-ionotropic NMDARs are dysregulated in the mouse model for Fragile X syndrome, thus by using proteomic and transcriptomic approaches. This will be done in the Host laboratory during the second phase of this MSC Action.
Then, using pharmacological approaches, we probed for the mode of signalling of involved in these two forms of synaptic plasticity. We revealed that timing-dependent long-term depression relies on non-ionotropic presynaptic NMDAR signalling (Figure Summary) whereas timing-dependent long-term potentiation relies on ionotropic postsynaptic NMDAR signalling. We have then deciphered further the signalling cascade mediating timing-dependent long-term depression. Using transgenic lines combined with pharmacology, we showed that non-ionotropic NMDARs determine timing-dependent long-term depression by recruiting the presynaptic JNK2 protein, which interacts with Syntaxin1a (Figure Summary). We have also demonstrated that this signalling pathway is required for timing-dependent long-term depression, regardless of the neuronal firing frequency during the induction (Figure Summary). Overall, our results show that NMDARs may have a specific mode of operation, depending on their synaptic localization as well as their signaling mode, to regulate specific forms of synaptic plasticity.
We now aim at testing whether these mechanisms are dysregulated in mouse model for Fragile X syndrome. In this line, we have started to study the main mouse model for Fragile X syndrome, the Fmr1 KO mouse line. Characterization is ongoing, and our preliminary data on synaptic plasticity have not led to any significant conclusions. Another step is to verify whether the signalling pathways downstream non-ionotropic NMDARs are dysregulated in the mouse model for Fragile X syndrome, thus by using proteomic and transcriptomic approaches. This will be done in the Host laboratory during the second phase of this MSC Action.
The results obtained so far in this project demonstrate that we have succeeded in discovering a new mode of NMDAR signaling in synaptic plasticity. This project also introduces a novel concept that has not been explored before: the involvement of unconventional NMDAR signaling in the pathophysiology of intellectual disability and autism, the two major cognitive disabilities associated with Fragile X syndrome. Studying this novel view of NMDAR signaling refines the current representation of NMDAR function and partners. During the second phase of this METAFRAX project, we aim to identify the specific molecular candidates that are dysregulated in Fragile X syndrome. Ultimately, a better understanding of how NMDAR signal in various forms of synaptic plasticity and how this is compromised in intellectual disability and autism will lead to the development of more targeted drug therapies, with fewer off-targets, and therefore less toxicity and side effects.
In addition, we characterized a novel mode of signalling of NMDARs using a combination of complimentary state-of-the-art techniques. These experiments are technically demanding and, to successfully obtain the results, we had to overcome the existing limitations of such technology., There is therefore no doubt these results will have an imminent impact on the scientific community, i.e. they could be subject of a large number of citations.
This collaborative research is key to public health because it will clarify the molecular mechanisms that underpin inherited intellectual disability and autism in Fragile X syndrome. The long-term goal is to enable the use of a refined NMDAR signaling pathway to develop and validate new pharmacotherapies based on protein-NMDAR interactions for the treatment of inherited intellectual disability and autism, in which NMDAR dysfunction drives the pathogenesis. These results, therefore, have important implications for the development of preclinical studies for pharmacotherapies for these brain diseases.
Ultimately, these preventive treatment or compensatory strategies will help improve cognitive function in individuals with inherited intellectual disability and autism, thereby ameliorating their quality of life. Therefore, this original project has the potential to be i) a revolutionary fundamental and therapeutic advance for several scientific fields, such as neurobiology and biomedicine, ii) of commercial interest to industries working for academic science or medical customers.
Finally, these results will certainly have a clear impact on the society. The dissemination of these results and the introduction of this new therapeutic strategies to the general public are bound to have a major societal impact. Thus, our current efforts to disseminate these results will undoubtedly payoff in achieving our scientific goals, as they will attract the attention of members of the scientific, commercial, medical and cultural communities.
In addition, we characterized a novel mode of signalling of NMDARs using a combination of complimentary state-of-the-art techniques. These experiments are technically demanding and, to successfully obtain the results, we had to overcome the existing limitations of such technology., There is therefore no doubt these results will have an imminent impact on the scientific community, i.e. they could be subject of a large number of citations.
This collaborative research is key to public health because it will clarify the molecular mechanisms that underpin inherited intellectual disability and autism in Fragile X syndrome. The long-term goal is to enable the use of a refined NMDAR signaling pathway to develop and validate new pharmacotherapies based on protein-NMDAR interactions for the treatment of inherited intellectual disability and autism, in which NMDAR dysfunction drives the pathogenesis. These results, therefore, have important implications for the development of preclinical studies for pharmacotherapies for these brain diseases.
Ultimately, these preventive treatment or compensatory strategies will help improve cognitive function in individuals with inherited intellectual disability and autism, thereby ameliorating their quality of life. Therefore, this original project has the potential to be i) a revolutionary fundamental and therapeutic advance for several scientific fields, such as neurobiology and biomedicine, ii) of commercial interest to industries working for academic science or medical customers.
Finally, these results will certainly have a clear impact on the society. The dissemination of these results and the introduction of this new therapeutic strategies to the general public are bound to have a major societal impact. Thus, our current efforts to disseminate these results will undoubtedly payoff in achieving our scientific goals, as they will attract the attention of members of the scientific, commercial, medical and cultural communities.